Excessive activity of neutrophils has been linked to many pathological conditions, including rheumatoid arthritis, cancer and Alzheimer's disease. Calpain-I is a Ca2+-dependent protease that plays a key role in the extravasation of neutrophils from the blood stream prior to causing damage within affected tissues. Inhibition of calpain-I with small molecule mercaptoacrylic acid derivatives slows the cell spreading process of live neutrophils and so these 'compounds represent promising drug leads. Here we present the 2.05 and 2.03 angstrom co-crystal X-ray structures of the pentaEF hand region, PEF(S), from human calpain with (Z)-3-(4-chloropheny1)-2-mercaptoacrylic acid and (Z)-3-(5-bromoindo1-3-y1)-2-mercaptoacrylic acid. In both structures, the alpha-mercaptoacrylic acid derivatives bind between two alpha-helices in a hydrophobic pocket that is also exploited by a leucine residue of the endogenous regulatory calpain inhibitor calpastatin. Hydrophobic interactions between the aromatic rings of both inhibitors and the aliphatic residues of the pocket are integral for tight binding. In the case of (Z)-3-(5-bromoindo1-3-y1)-2-mercaptoacrylic acid, hydrogen bonds form between the mercaptoacrylic acid substituent lying outside the pocket and the protein and the carboxylate group is coplanar with the aromatic ring system. Multiple conformations of (Z)3-(5-bromoindo1-3-y1)-2-mercaptoacrylic acid were found within the pocket. The increased potency of (Z)-3-(5-bromoindo1-3-y1)-2-mercaptoacrylic acid relative to (Z)-3-(4-chlorophenyI)-2-mercaptoacrylic acid may be a consequence of the indole group binding more deeply in the hydrophobic pocket of PEF(S) than the phenyl ring. (c) 2014 Elsevier Inc. All rights reserved.